Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-7: 5002-5009

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Special Issue-7 pp. 5002-5009 Journal homepage: http://www.ijcmas.com

Review Article

Cisgenesis A Sustainable Approach of Gene Introgression and Its Utilization in Horticultural Crops: A Review

Vaibhav Singh1*, Saurabh Singh1, Kumari Shikha2 and Ashutosh Kumar1

1Horticulture; I.Ag.Sc., Banaras Hindu University, India 2Genetics and ; I.Ag.Sc., Banaras Hindu University, India *Corresponding author

ABSTRACT

Plant breeding is on-going process for which heritability of trait and variation is required. Cisgenesis and Intragenesis are biotechnological approaches utilized to create more genetic variation in existing germplasm with the purpose to improve their quality and quantity. The

advancement of sequencing technologies and the availability of genome information

facilitates isolation of intact Cis-genes, together with associated promoter/terminator from K e yw or ds one species and are inserted into the genome of the same or a closely related, crossable species. While, in case of intragenesis different coding and regulatory sequences are Cisgenesis, assembled either in sense or in antisense orientation. This review describes the current Intragenesis, status, historical overview, applications, limitations and future prospects of Cisgenesis and Heritability, Intragenesis in improvement of horticultural crops. Further comparative study of Linkage drag, Plant breeding and transgenesis, cisgenesis and intragenesis described. This technique generally utilized to Resistance develop disease resistance cultivar such as late blight resistant potato by transferring gene Rpi-sto1, Rpi-vnt1.1; similarly scab resistance apple cultivar developed. Thus we can conclude Cisgenesis as a powerful alternative approach to transfer gene of interest without

linkage drag in a single-step and Cisgenic & intragenic derived genetically modified plants (GMP) are eco-friendly as classic bred plants and therefore exempted from GMP

legislation.

Introduction

Plant breeding is a long-term process, often genetic variation present in unrelated takes more than 10 years to develop a species. Plant diversity is the fundamental variety especially for a tree crop like apple. for the future horticulture industry to buffer Therefore there is urgent need to employ insect, disease and abiotic stresses and biotechnological tools to mitigate abiotic develop better quality products [2]. We can and biotic stress by developing resistant and get the desired gene from existing improved quality varieties of vegetables, germplasm or they can be recombined to fruits and ornamental crops that can survive isolate novel trait. Transgenic plants (GMP) well in future. Advances in plant molecular have shown promising result in developing and bioinformatics furnishes the tolerance to abiotic and biotic stress. But, isolation of plant genes associated with commercialization and consumption of economically important traits, [1] which transgenic horticulture crops lagged behind provides alternative approaches to on by agronomic crops due to involvement of 5002

Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-7: 5002-5009 environmental and health risk. To overcome DNA pool” called P-DNA (instead of T- limitations associated with utilization of DNA) borders [5].Availability of genome and transgenic horticultural crops, new sequencing information facilitates all these biotechnological plant technology (NBPT) steps in achieving Cisgenic/Intragenic lead to advancement of new eco-friendly plants. Erstwhile, -mediated and speedy approaches such as cisgenesis transfer of a gene from a crossable (sexually and intragenesis. In cisgenesis and compatible) plant where T-DNA borders intragenesis breeding plants must be may remain in the resulting organism after transformed with genetic material derived transformation cisgenesis with T-DNA from the species itself or from closely borders also called cisgene[6]. While, in case related species which are capable of sexual of intragenesis concept P-borders and hybridization. This new technique is in vector-backbone sequences do not raise contrast to transgenesis where genes and from the sexually compatible DNA pool [7]. DNA sequences translocate between any Even, intragenic plants do not contain species. foreign genes such as selectable marker genes or insecticidal genes. Also gene Evert Jacobsen and Henk Schouten are silencing techniques may be utilized, e.g. considered as the 'fathers' of cisgenesis and through RNAi[8]. Schouten coined the term „cisgenesis‟ in 1999. The principle of cisgenesis entails that However, the definitions of cisgenesis and the genes or gene elements should be intragenesis should be more precise with derived from the species itself, while there respect to the regulatory requirements for were no requirements for the coding Cisgenic and intragenic crops in the future sequence to include introns or for the will be subjected to less stringent regulatory regulatory sequences to originate from the procedures compared to transgenic crops. same gene as the coding sequences. The Likewise, it will be important that definition of cisgenesis introduced by Dutch international definitions are harmonized to Scholars Schouten, Jacobsen and Krens prevent difficulties for the global trade of which was internationally accepted when these products. Overall, we can say published in international journals in 2006[3]. thatIntragenesis and cisgenesis exploit According to this concept the origin of the similar genepool as traditional breeding cisgene is extended to the gene pool of does. Hence,cisgenic crops considered more sexually compatible species and the cisgene natural/acceptable than transgenic crops include its promoter, introns and the [9].Furthermore, some modification may help terminator in the normal-sense orientation.In to develop marker and vector-backbone production of Cisgenic derived plants, gene free intra-genic/cisgenic transformants isolation of gene, and transformation or their progeny. As production of marker- are important steps. When the is free transgenic crops eliminates risk used to insert gene cassettes into a host crop ofhorizontal gene transfer and could genome, the concepts are easier to follow mitigate vertical genetransfer[10]. because only the gene cassettes can be inserted into the host genome [4]. However, Need for cisgenesis when Agrobacterium DNA delivery system is utilized for modification of a crop In case of conventional breeding genome, then intragene must be inserted introgression of foreign gene from related or “within borders isolated from the crossable unrelated species through backcross

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Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-7: 5002-5009 breeding needs almost 8-10 years. While One of the main requirements is the absence backcrossing there is probability of transfer of selectable marker genes (such as of undesirable alleles along with desirable antibiotic resistance genes) in the genome. one due to tight linkage between them this phenomenon called linkage drag. The sensitive issues with regard to transfer of foreign genes and antibiotic resistance are Such limitations of classical breeding can be overcome by employing such advanced overcome by employing cisgenesis and biotechnological tool [14]. intragenesis approach. Comparison between cisgenesis, Vegetatively propagated crops such as intragenesis, transgenesis and potato, banana, apple etc. are heterozygous conventional breeding (polyploid) in nature, so introgression of desired gene or gene stacking is difficult So far, we reviewed that cisgenic plants are through conventional breeding. There are similar to conventional products as extra two ways to improve the existing varieties gene derived from the same gene pool of the with, i.e. (induced) mutation breeding and recipient parent and also contain genes and genetic modification. Mutation breeding is regulatory elements in their native state. still followed in vegetatively propagated However, some differences exist that ornamentals that are heterozygous like rose, distinguish cisgenesis from conventional Chrysanthemum and in fruit trees like apple breeding as cisgenesis modify target gene and peach[11]. But in few crops like potato, while in classical breeding there is application of mutation breeding found to possibility of contamination by undesirable have adverse effect on traits like tuber skin gene. Also, traditional breeding take decades colour and other tuber related traits. In that to reach a cultivar with desirable traits while case, cisgene and intragene would be useful cisgenesis furnishes fast, safe and acceptable to avoid such hazard from unidentified hitch introgression of desired gene. For e.g. hiking genes [12]. Introgression of apple scab resistance gene Vf from Malus floribunda 821 into Higher expression levelof a trait can be marketable high quality apple cultivars took obtained by re-introducing the gene of the approximately 50 years through classical traitwith its own promoter and terminator breeding[15].The fundamental differences (cisgenesis) or with apromoter and between cisgenic, intragenic, transgenic terminator isolated from the sexually from conventional breeding are listed in compatiblegene pool (intragenesis). Lower Table 1. expression levels can be obtainedthrough different silencing constructs (intragenesis). Application of cisgenesis in crop improvement From safety point of view, Cisgenic plants are considered non-transgenic as genes There are several vegetables, fruits and within the same gene pool transferred into ornamental plants which were modified novel varieties without undesirable genes through cis-intragenesis technique. and alien gene. It is random process, similar However, it is restricted to few crop species. to traditional induced translocation breeding Few examples discussed where these new [13]. As a result such plants can be introduced approach have been exploited to improve into the food chain without any regulation. specific defects in crop.

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Cisgenic melon plants developed by and carotenoids[21]. In particular, these transforming genes At1 and At2 from wild technologies represent possible alternatives melon to susceptible variety resulted in for increasing the accumulation of enhanced activity of glyoxylate commercially relevant metabolites through aminotransferase and resistance against the overexpression of native enzymes, in powdery mildew (P. cubensis) [16]. accordance with the needs of consumers and in line with environmental conservation [22]. Acetolactatesynthase gene utilized as a cisgenic selectable marker for These new breeding technologies can lead Agrobacterium-mediated transformation in not only to cisgenic plants but also to Chinese cabbage (Brassica marker-free transgenic plants[23]. rapassppekinensis)[17]. Cisgenesis may furnish the second green Cisgenic apple plants resistant to scab revolution in India by improving traditional diseasedeveloped by transferring the Rvi6 plant breeding [24]. Cisgenesis has great scab resistance gene of Malus floribunda potential to overcome bottlenecks of 821, using a new transformation vector classical breeding and transgenesis. based on the Flp/FRT recombinase system in Currently, plant breeders and researchers cultivated cultivar[18]. objective is to create genetic variability, improve quality and to breed for stress A cisgenic apple line C44.4.146 developed tolerance and disease and pest resistance from susceptible cultivar “Gala Galaxy” (plant incorporated protection, PIP) in using the cisgene FB_MR5 from wild apple adapted cultivar by incorporating additional Malus × robusta 5 (Mr5), which confers copies of a given gene. This gene resistance to fire blight[19]. modification implemented in several important crops like cereal crops, legumes, Cisgenesis is an acceptable tool to make root plants, vegetables, turf grasses, tropic potato farming more sustainable by plants, woody species, medicinal and developing durable late blight resistant ornamental plants, as well as fruit plants variety of potato. Late blight resistance (R) [25,26]. genesfrom crossable wild potato species cloned and transferred to susceptible cultivar Recently, new cisgenic/intragenic and by Agrobacterium tumefaciens-mediated genome editing approaches based on the transformation without non-potato genes[20]. advancement of sequencing technologies A stack of multiple R genes were inserted and the availability of genome information into established varieties, thereby creating a facilitates the exploitation of PGR in crop dynamic variety in which the composition of breeding [27]. the stacks may vary over space and time. Cisgenic plants were selected based on the Limitations expression of all inserted R genes and trueness-to-type. The major bottleneck in cisgenesis may be identification of genes encoding for the Cis-intragenesis utilized in microalgae for desired traits and detailed experimental commercial extraction and production of characterisation required for the efficient use attractive and highly valued compounds, of identified genes, which is cost- and time- particularly ofpolyunsaturated fatty acids consuming.

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Table.1 Factors that make cisgenesis and intragenesis different from transgenesis and conventional breeding

Characters Conventional Transgenic Cisgenic Intragenic breeding 1. Transfer of Not possible Done from Done between Done between crossable unique gene unfamiliar species crossable related related species species 2. Nature of Natural gene Foreign gene may Natural gene (Gene Natural gene (Gene of gene be natural or of interest including interest from one source artificial its own regulatory and include regulatory elements and introns) elements and introns from other source) 3. Speed Slow Rapid Rapid Rapid 4. Time- Long-term process Single- step process Single- step process Single- step process period requirement 5. Marker Not removed Not removed Removed Removed gene 6. Eco- Yes No Yes Yes friendly 7. Freefrom Yes No Yes Yes GMP legislations 8. Change in No Yes, widen the No No gene pool genetic resources 9. Possibility Yes Yes No No of linkage drag

For effective expression, cisgene should get depends on the method applied and needs to inserted at active region of gene but there is be verified individually. possibility to get inserted at random places of the host plant genome, which could alter Native promoters may lead to constitutive the expression of the same gene or of expression of genes, which may be above adjacent genes, thus producing variations the native expression level of a gene, as that are difficult to predict [28]. shown for the HcrVf2 gene conferring apple scab resistance[30]. The altered expression Transfer of polygenic trait is difficult can alter the environmental behaviour of the through this technique. So this technique plant and also furnish considerations restricted to improve monogenic traits. concerning exposure of potential consumers necessary. Gene silencing is not possible To ensure that new resistances are not through cisgenesis can be done by utilizing broken rapidly, a combination of genes intragene. Silencing of unwanted genes like should be inserted into the recipient plant gene involved in biosynthesis of asparagine (gene stacking, multi-gene cassettes). in potato attempted by Rommens et al., [31] and Chawla et al., [32]. Plant transformation remains a tedious procedure; particularly in fruit trees[29]. The Exemptions from GMO legislations same limitations (e.g. random integration of genetic constructs) as for transgenic plants So far, there is stiff public opposition to the apply. The complete removal of selection use of genetically modified organisms markers is indispensable; the efficiency (GMOs) or plants (GMPs). But, still several

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